L-Serine is a key intermediate in a number of important metabolic pathways. It is an essential or major precursor of taurine and L-cystathionine, of sphingosine and therefore of spingomyelin and the complex sphingolipids; of certain classes of phospholipids, of porphyrins, purines, thymidine, and of the one-carbon pool and the methylation reactions. Therefore, L-serine has key roles in both the structural elements of the cell and the metabolic regulation of important pathways. The project is a basic study of the kinetics and thermodynamics of mammalian L-serine biosynthesis using both in vitro and in vivo approaches. The overall goal of the current proposal is to define the short-term regulation of L-serine biosynthesis in vivo using rabbit liver as an animal model. The project may be subdivided into two broad areas: 1) Enzyme studies, which include the purification and detailed kinetic analysis of the forward and reverse reactions of the enzymes of the phosphorylated pathway of L-serine synthesis (D-3-phosphoglycerate dehydrogenase, L-phosphoserine aminotransferase, and L-phosphoserine phosphatase); and 2) Animal model studies, which include the use of hepatocytes, perfused liver and intact animals to investigate short-term regulation of the pathway during conditions of differing rates of L-serine biosynthesis. Techniques and approaches include i) enzyme purification and kinetic analysis, ii) measurement of the rate of L-serine (+ glycine) accumulation under conditions of varying L-serine synthesis, iii) determination in tissue of intermediates and effectors of the pathways of L-serine biosynthesis by a variety of enzymatic procedures, and iv) validation of radiotracer precursor studies in vivo. The combination of these techniques in a single animal model offers to supply a very complete description of a metabolic pathway and its regulation.